This invention provides a method to permit optical detection of localised calcium signaling (e.g. high Ca2+ concentration microdomains) using a genetically encoded bioluminescent reporter. This invention describes a method to detect the effect of a pharmacological agent or neuromodulator on localised Ca2+ signalling. The invention especially provides a method to visualise dynamic fluctuations in localised Ca2+ associated with cell or tissue activation, such as neuronal activation and relating to optical detection of ion channel function (receptors/channels permeable to Ca2+) and synaptic transmission. This invention also concerns a method for optical detection of the dynamics of Ca2+ in a biological system, said method comprising monitoring the photons emitted by a recombinant Ca2+-sensitive polypeptide, which comprises or consists of a chemiluminescent protein linked to a fluorescent protein, present in said biological system. Also, this invention provides a transgenic non-human animal expressing a recombinant polypeptide sensitive to calcium concentration, consisting of at least a chemiluminescent protein linked to a fluorescent protein, in conditions enabling the in vivo monitoring of local calcium dynamics. Ca2+ is one of the most universal and physiologically important signaling molecules that plays a role in almost all cellular functions, including fertilization, secretion, contraction-relaxation, cell motility, cytoplasmic and mitochondrial metabolism, synthesis, production of proteins, gene expression, cell cycle progression and apoptosis (Rizzuto et al., 2002).
Characteristics of Ca2+ transients at the cellular and subcellular level are complex, and vary according to spatial, temporal and quantitative factors. Up to a 20,000-fold difference in the concentration of Ca2+ exists between the cytoplasm and the extracellular space, such that even when channels are open for a short time, a high rate of Ca2+ influx will occur. Factors such as diffusion, Ca2+ binding to buffer proteins and sequestration by cellular compartments, will create a Ca2+ gradient and result in a high concentration microdomain within a few hundred nanometers from the pore of a channel. Over longer distances such as tens of microns, the effective diffusion coefficient of Ca2+ will be strongly reduced.
Because Ca2+ signals are highly regulated in space, time and amplitude, they have a defined profile (e.g. amplitude and kinetics). Ca2+ transients are shaped by cytosolic diffusion of Ca2+, buffering by Ca2+ binding proteins and Ca2+ transport by organellar (Bauer, 2001; Llinas et al., 1995). The concentration of Ca2+ reached and its kinetics in any given cellular microdomain is critical for determining whether a signaling pathway succeeds or not in reaching its targets. Ca2+ is necessary for activation of many key cellular proteins, including enzymes such as kinases and phosphatases, transcription factors and the protein machinery involved in secretion. Ca2+ signaling cascades may also mediate negative feedback on the regulation of biochemical pathways or functional receptors and transport mechanisms. The propagation of Ca2+ within a cell can also help to link local signaling pathways to ones that are more remote within a cell or for facilitating long distance communication between cells or networks of cells (e.g. central nervous system) (Augustine et al., 2003).
Ca2+ transients producing high Ca2+ concentration microdomains are associated with a diverse array of functions important in development, secretion and apoptosis, and many cellular processes, including gene expression, neurotransmission, synaptic plasticity and neuronal cell death (Augustine et al., 2003; Bauer, 2001; Llinas et al., 1995; Neher, 1998). Characterising the spatiotemporal specificity of Ca2+ profiles is important to understand the mechanisms contributing to perturbed cellular Ca2+ homeostasis, which has been implicated in many pathological processes, including migraine, schizophrenia and early events associated with the onset of neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases (Mattson and Chan, 2003). Because Ca2+ is directly or indirectly associated with almost all cell signaling pathways, optical detection of Ca2+ is a universal measure of biological activity at the molecular, cellular, tissue and whole animal level. Tremendous progress has been made in the imaging of localised Ca2+ events using light microscopy. To this end, Ca2+ signalling in single dendritic spines (Yuste, 2003) and more recently in a single synapse (Digregorio, 2003) has been accomplished using fluorescent dyes. However, one way to spatially improve measurements of Ca2+ is to genetically target a reporter protein to a specific location whereby Ca2+ activity can be directly visualised. Specifically, such a reporter protein could be fixed in a microdomain (within 200 nm of the source or acceptor) or even within a nanodomain (within 20 nm) (see Augustine et al. 2003 for review). Expression of a reporter gene under the control of cell type-specific promoters in transgenic animals, can also offer a non-invasive way to follow dynamic changes in a single cell type, tissues or anatomically in whole animal imaging.
Monitoring calcium in real-time can help to improve the understanding of the development, the plasticity and the functioning of a biological system, for example the central nervous system. Indeed, much effort has been dedicated to the development of an optical technique to image electrical activity in single-cell type and particularly single neurons and networks of neurons, but there continues to be a need to achieve this goal through use also of electrophysiological techniques. Genetic targeting of a Ca2+ reporter probe in spatially restricted areas of a cell or living system (e.g. inside of a compartment, to microdomains or nanodomains, or by fusion to a specific polypeptide) is a molecular imaging approach for detecting specific cellular activities or physiological functions. This invention aids in fulfilling these needs in the art, by providing a method for optical detection of the dynamics of Ca2+ in a biological system, said method comprising monitoring the photons emitted by a recombinant Ca2+-sensitive polypeptide, which comprises or consists of a chemiluminescent protein linked to a fluorescent protein, present in said biological system, as well as a transgenic non-human animal expressing said recombinant polypeptide sensitive to calcium. The non-invasive nature of this technique as well as the evidence that the recombinant protein is non-toxic, means that the method could possibly also be applied in humans.